Internal polyadenylation of parvovirus RNA is more prevalent than previously appreciated, and in fact, can be considered the rule rather than the exception as a means to govern parvovirus gene expression. It can occur either within or outside of a functional intron, but in all cases internal polyadenylation precludes extension into the virus capsid-coding gene. Thus, it is a key facet of parvovirus gene expression, and understanding its mechanism and impact are critical. Although all AAV5 RNAs have the same single intron, the relative rates of splicing vs internal polyadenylation at (pA)p varies and depends on the size of the 5'-exon, i.e., the distance between the RNA initiation site and the intron donor. Our initial characterization of AAV5 internal polyadenylation has led us to propose a model in which the distance-dependent processing of AAV5 RNA is controlled by the strength of U1snRNP binding to the nonconsensus intron donor in a manner governed by 5'-exon definition, via interaction with the nuclear cap-binding complex (CBC) bound to the RNA cap site. This model can also explain the distance-dependent splicing of AAV2 RNA. In the first two specific aims of this application, we propose to critically test the two major steps of our model, namely: i) how the size of the AAV5 and AAV2 5'-exon governs U1snRNP binding to the intron donor; and ii) how U1snRNP inhibits polyadenylation at AAV5 (pA)p. In the third specific aim we will determine the genetic differences that allow AAV5, but not AAV2, to internally polyadenylation, and determine how such differences impact their life cycles. In the fourth specific aim we will begin to develop a new model. We will characterize internal polyadenylation of RNA generated by the Erythrovirus B19, the only parvovirus other than AAV5 and the non-primate dependoviruses, to utilize internal polyadenylation within a functional intron. Knowledge gained from the proposed studies will provide important insight both into a critical aspect of parvovirus gene expression, and into the mechanism of exon definition in general. And, as has been true in the past, detailed studies of parvovirus molecular genetics will continue to provide attractive, tractable model systems for studying basic molecular mechanisms of gene expression. PUBLIC HEALTH RELEVANCE: Parvoviruses are small (20nm) non-enveloped icosahedral viruses that infect and cause disease in many vertebrate hosts. They are also highly attractive vehicles for gene therapy applications. They are unique among all known animal viruses in that they contain single- stranded linear DNA genomes. They have a compact genetic organization featuring overlapping transcription units which utilize extensive alternative splicing, alternative polyadenylation, and alternative translation initiation. We have begun to characterize the mechanism of alternative polyadenylation of parvovirus RNA at the molecular level. We propose to expand this analysis in this proposal. The knowledge gained from our studies will advance our understanding of this important mechanism of gene expression in this important group of viruses.